A new microscope with novel capabilities to advance our studies of cell adhesion, signal transduction and cytoskeletal regulation

描述(申请人提供)：协调细胞黏附和细胞骨架在发育和动态平衡过程中对塑造组织和器官至关重要。我们建立了一个模型，用高度多学科的方法研究果蝇形态发生过程中的这种情况。我们关注的蛋白质在哺乳动物发育和癌症转移中也起着关键作用。因此，了解黏附和细胞骨架的协调机制可以促进基础科学和临床应用。在确定了介导黏附和调节肌动蛋白的蛋白质后，我们面临着一个新的挑战：细胞如何利用这些分子机器来执行不同的细胞行为塑造发育。在过去的四年里，我们确定了Canoe/afadin在四个中央发育事件中作为黏附连接(AJ)-肌动蛋白连接物的角色，并开始将其置于调节形态发生的蛋白质网络中。我们还确定了两个关键的肌动蛋白调节因子：Ena/Vasp蛋白和Dia类福尔马林在形态发生中的作用。我们发现Ena和Dia以复杂的、非相加的方式在物理上相互作用和功能协作，并探索Abelson激酶如何整合上游信号和协调多个肌动蛋白调节因子的活性。这些数据是我们探索新的细胞生物学范式的目标的基础。目的1确定RAP1和CANOE以及它们的相互作用分子网络如何共同和单独地调节顶底极性和柱状细胞形状的建立和维持。我们的数据显示，Canoe和Rap1是形态发生过程中AJ：肌动蛋白连接的关键调节因子，并表明它们在细胞极性的启动和维持中发挥着意想不到的作用。我们的工作假设是，它们这样做是作为调控网络的一部分，包括AJ蛋白、顶极性蛋白Bazooka/Par3、肌动蛋白细胞骨架和未知的效应器。我们将在两个关键事件中定义它们是如何单独和共同工作的：顶端-基底端极性的建立和维持以及柱状细胞形状的建立。这一目标的成功完成将为连接蛋白、细胞骨架蛋白和极性蛋白的重叠网络如何控制不同的细胞行为提供新的机制见解。目的2明确肌动蛋白在形态发生过程中的协调调控机制。我们的数据表明，Dia和Ena以非相加的方式协同调节肌动蛋白，而Abl是整合多个细胞骨架调节因子的支架。我们的工作假设是，直接的Ena：Dia相互作用调节它们在带刺末端的作用，产生不同的细胞行为，Abl通过多个部分冗余的相互作用充当支架，并使用Ena和Abi作为效应器。我们将使用多学科的方法来研究Ena和Dia在体外、模型细胞和胚胎中的合作，并将定义Abl和效应器如何协调调节细胞骨架。这一目标的成功完成将为肌动蛋白调节器如何通过物理相互作用进行合作以及如何由支架蛋白协调调节以产生具有不同动力学和功能的肌动蛋白结构来驱动形态发生提供新的机制见解。

DESCRIPTION (provided by applicant): Coordinating cell adhesion and the cytoskeleton are critical for shaping tissues and organs during development and homeostasis. We established a model to study this during Drosophila morphogenesis, using a highly multidisciplinary approach. Proteins on which we focus also play key roles in mammalian development and cancer metastasis. Understanding mechanisms coordinating adhesion and the cytoskeleton thus advances both basic science and clinical applications. Having identified proteins mediating adhesion and regulating actin, we face a new challenge: how do cells use these molecular machines to carry out the diverse cell behaviors shaping development. In the last four years we defined roles for Canoe/afadin as an adherens junction (AJ)-actin linker in four central developmental events and began to place it in a protein network regulating morphogenesis. We also defined roles in morphogenesis of two key actin regulators: Ena/VASP proteins and Dia-class formins. We found Ena and Dia physically interact and functionally cooperate in complex, non-additive ways, and explored how Abelson kinase integrates upstream signals and coordinates activity of multiple actin regulators. These data underlie our Aims, which explore new cell biological paradigms. Aim 1 Determine how Rap1 and Canoe and their network of interactors work together and separately to regulate establishment and maintenance of apical-basal polarity and columnar cell shape. Our data reveal that Canoe and Rap1 are critical regulators of AJ:actin linkage during morphogenesis, and demonstrate they play unexpected roles in initiation and maintenance of cell polarity. Our working hypothesis is that they do so as part of a regulatory network including AJ proteins, the apical polarity protein Bazooka/Par3, the actomyosin cytoskeleton, and unidentified effectors. We will define how they work individually and together in two key events: apical-basal polarity establishment and maintenance and establishing columnar cell shape. Successful completion of this Aim will provide novel mechanistic insights into how diverse cell behaviors are controlled by overlapping networks of junctional, cytoskeletal and polarity proteins. Aim 2 Define mechanisms by which actin dynamics are coordinately regulated during morphogenesis. Our data suggest Dia and Ena cooperatively regulate actin in non-additive ways, and Abl is a scaffold integrating multiple cytoskeletal regulators. Our working hypothesis is that direct Ena:Dia interaction modulates their action at barbed ends, producing diverse cell behaviors, and that the Abl acts as a scaffold via multiple partially redundant interactions, and uses both Ena and Abi as effectors. We will use a multidisciplinary approach to study Ena and Dia cooperation in vitro, in model cells, and in embryos, and will define how Abl and effectors coordinately regulate the cytoskeleton. Successful completion of this Aim will provide novel mechanistic insights into how actin regulators cooperate via physical interaction and how they are coordinately regulated by scaffolding proteins to produce actin structures with distinct dynamics and functions to drive morphogenesis.